Structure-guided simulations illuminate the mechanism of ATP transport through VDAC1

Abstract

The voltage-dependent anion channel (VDAC) mediates the flow of metabolites and ions across the outer mitochondrial membrane of all eukaryotic cells. The open channel passes millions of ATP molecules per second, whereas the closed state exhibits no detectable ATP flux. High-resolution structures of VDAC1 revealed a 19-stranded β-barrel with an α-helix partially occupying the central pore. To understand ATP permeation through VDAC, we solved the crystal structure of mouse VDAC1 (mVDAC1) in the presence of ATP, revealing a low-affinity binding site. Guided by these coordinates, we initiated hundreds of molecular dynamics simulations to construct a Markov state model of ATP permeation. These simulations indicate that ATP flows through VDAC through multiple pathways, in agreement with our structural data and experimentally determined physiological rates.

Fig. 1. Cartoon representations of the refined mVDAC1 in complex with ATP

mVDAC1 in complex with ATP viewed from within the plane of the membrane (left) and from the cytosol (right). ATP is bound to mVDAC1 at the center of the pore, forming hydrogen bonds between the side chain nitrogens of residues Lys12 and Lys20 with the O2γ and O1γ of the phosphate tail (2.3 and 3.3 Å, respectively). A structural water (magenta) interacts the N6 nitrogen on the adenosine ring (N6-water oxygen distance is 3.2 Å), and the water also hydrogen bonds to Lys256 Nζ and Glu280 Oε2 (distances to water oxygen are 2.7 and 2.9 Å, respectively). The polypeptide chain is in cartoon representation with the β-barrel and the N-terminal α-helix segment colored cyan and red, respectively. All highlighted interactions are indicated by black dashed lines. The side chains and ATP are shown in ball-and-stick representation. To facilitate visualization of ATP, strands 7–10 are partially transparent. The inner pore dimensions are indicated in the left panel.

Fig. 2. Simulated current-voltage curves and ion permeation rates for mVDAC1

a–c, Current-voltage curves under different ionic and ATP conditions. The current carried by K⁺ is represented by red circles, the Cl⁻ current is represented by blue squares and the total current is their sum represented by black diamonds. The lines are linear regressions of the respective data points, and they represent the current-voltage curves for the total current (black line), Cl⁻ current (blue line) and K⁺ current (red line). Panel a is compiled from 16.9 μs of aggregate simulation time carried out on a system in 142 mM KCl with ATP in the channel pore. Panels b and c are compiled from shorter 60 ns simulations under high salt (900 mM KCl) with or without ATP in the pore, respectively. d–f, The cumulative net number of channel crossing events by Cl⁻ (blue traces) and K⁺ (red traces) tracked over the time course of each simulation at +50 mV for physiological salt (142 mM) in d, high salt (900 mM) with ATP in the pore in e, and high salt (1 M) with no ATP in the pore in e.

Fig. 3. ATP adopts many conformations in the mVDAC1 pore

a, Steady state distribution of ATP in the pore domain, defined as −18 Å ≤ z ≤ 20 Å, calculated from the Markov State Model (red). The most stable state (#73) has 16% occupancy. The data from the longest Anton simulation (4.8 μs) was projected onto the MSM states (blue), and the time spent in each state was used to determine the relative probability of occupancy (plotted in the negative direction for clarity). b, The most stable ATP configurations are structurally diverse. The generators for the 1^st (#73, blue), 2^nd (#77, green), and 9^th (#22, red) most populated states are depicted. The phosphate tails of states 73 and 77 interact with basic residues (Lys12, Arg15 and Lys20) on the N-terminal helix, while the phosphate tail of state 22 interacts with Lys113 and Lys115 on the wall of the β-barrel.

Fig. 4. Comparison between experimental ATP structure and MSM configurations

Overlay of the X-ray structure with ATP configurations taken from the top three most structurally similar MSM states: a, state 58 (19^th most populated), b, state 148 (10^th most populated) and c, state 73 (1^st most populated). In each panel, the ATP coloring scheme for the X-ray structure is as in Fig. 1, and each structure from the MSM is solid green. The RMSD value between the experimentally determined ATP molecule and the representative MSM configuration is shown in the corresponding panel.

Fig. 5. A high ATP flux is achieved through multiple, distinct pathways

a, Rank ordering of all distinct ATP flux pathways through the channel from the IMS to the cytoplasm as identified via transition path analysis. The highest flux pathways giving rise to 70% of the total flux were identified and grouped into 5 primary paths based on spatial analysis in panel b. Paths were numbered according to their probability and color coded. b, The γ-phosphate of ATP was plotted for all of the highest flux pathways identified in panel A. The arrow indicates the common entry point for all paths.

Fig. 6. ATP permeates via a network of basic residues

a, The highest probability pathway (34%) from the IMS to the cytoplasm moves up and over the N-terminal helix. In all panels, arrows indicate the direction of flow from the IMS to the cytosol, and * indicate the rate limiting steps. For paths 1–4, the rate-limiting step is release from the patch of basic residues on the helix (Lys12, Arg15 and Lys20). Solid lines delineate the membrane boundaries. b, Paths 2–4 (15%, 13% and 7% probability, respectively) share many states in common, except for the final state prior to escape to the cytoplasm. Numbers next to the final transition differentiate the paths. c, Path 5 (2% probability) is the least probable path, and it avoids the basic residues on the helix.